Identification of residues within UvrB that are important for efficient DNA binding and damage processing

Abstract

The UvrB protein is the central recognition protein in bacterial nucleotide excision repair. We have shown previously that the highly conserved β-hairpin motif in Bacillus caldotenax UvrB is essential for DNA binding, damage recognition, and UvrC-mediated incision, as deletion of the upper part of the β-hairpin (residues 97-112) results in the inability of UvrB to be loaded onto damaged DNA, defective incision, and the lack of stranddestabilizing activity. In this work, we have further examined the role of the β-hairpin motif of UvrB by a mutational analysis of 13 amino acids within or in the vicinity of the β-hairpin. These amino acids are predicted to be important for the interaction of UvrB with both damaged and non-damaged DNA strands as well as the formation of salt bridges between the β-hairpin and domain 1b of UvrB. The resulting mutants were characterized by standard functional assays such as oligonucleotide incision, electrophoretic mobility shift, stranddestabilizing, and ATPase assays. Our data indicated a direct role of Tyr96, Glu99, and Arg123 in damage-specific DNA binding. In addition, Tyr93 plays an important but less essential role in DNA binding by UvrB. Finally, the formation of salt bridges between the β-hairpin and domain 1b, involving amino acids Lys111 bound to Glu307 and Glu99 bound to Arg367 or Arg 289, are important but not essential for the function of UvrB.